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Y.E. Miller
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MS 14 - Chemo Prevention Clinical Trials (ID 32)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Prevention and Tobacco Control
- Presentations: 4
- Moderators:Y.E. Miller, P.E. Postmus
- Coordinates: 9/08/2015, 14:15 - 15:45, 401-404
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MS14.01 - Preclinical Models: How Good Are They? (ID 1908)
14:15 - 15:45 | Author(s): R.L. Keith
- Abstract
- Presentation
Abstract:
A commonly employed approach to understanding the pathogenesis of lung neoplasia is to use experimental animal models. The testing of potential chemopreventive (and chemotherapeutic) agents involves pre-clinical testing, and numerous animal models have been developed. In primary mouse lung tumor models, lung cancer develops through a predictable series of airway lesions that progress from normal epithelium to invasive cancer. Permanent genomic DNA alterations occur through either spontaneous, chemically, or environmentally-induced initiation events. The lung cancer chemoprevention field is increasingly reliant on animal studies as the results of negative, early, large scale human studies (for example, β-carotene) may have been predicted with extensive pre-clinical testing. Agents progressing to human trials now undergo extensive pre-clinical studies, and this review will focus on the commonly utilized models of adenocarcinoma (ADC) and squamous cell carcinoma(SCC). ADC: Multiple, well-characterized models of murine adenocarcinoma are available in which pulmonary adenomas progress to adenocarcinomas. These progression models also allow for the study of pre-malignant airway lesions. The most commonly studied models include initiator-promoter carcinogenesis(1), mutant KRAS(2) or EGFR(3) and the use of complete carcinogens. Urethane, a component of cigarette smoke, is a complete carcinogen because it leads to tumor development without the need for other carcinogens or promoters(4). 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) , another tobacco smoke carcinogen, is a chemically-induced model where the NNK is typically administered in drinking water or injected intraperitoneally. Tobacco smoke models also exist and they can reproducibly induce pulmonary adenocarcinomas, but they are fairly labor-intensive and do not result in robust tumor multiplicity(5). There are also multiple two-stage murine models of lung tumor promotion. Several of them use 3-methlycholanthrene (3-MCA), a polycyclic aromatic hydrocarbon, as the initiator at a low dose, followed by multiple doses of butylated hydroxytoluene (BHT). BHT is a well known antioxidant found in processed foods and packaging, however it is also an oxidant and well characterized promoter. In addition, genetically modified animal models, in which viral oncogenes or transforming ras mutants are selectively expressed in lung tissue, have been developed and extensively investigated. While there are differences between the human and murine respiratory tracts, the murine tumors derived from these models have many similarities to human adenocarcinoma, ranging from specific markers to gene expression patterns(6). SCC: Models of squamous cell lung cancer have also been developed, but they are much fewer in number. The most commonly utilized SCC model involves NTCU (N-nitroso-tris-chloroethylurea), which is applied topically and histopathological analysis of serial lung sections in this model revealed a range of lung pathology, including squamous-cell carcinoma, carcinoma in situ, and varying levels of bronchial dysplasia(7). Immunohistochemical studies on the premalignant lesions show staining that corresponds to analogous human lesions(8), and the NTCU model also induces dysplastic lesions that are similar to those found during bronchoscopy and can therefore be used to evaluate one proposed surrogate endpoint in pre-clinical studies (endobronchial dysplasia). Several positive murine chemoprevention studies examining ginseng, pomegranate fruit extract, and aerosolized budesonide +/- pioglitazone have also used NTCU (reviewed in (9)). Additional SCC models have been developed, the first consisting of a kinase dead IKKα knockin mouse (Ikkα[K44A/K44A], Ikkα[KA/KA]) that develop spontaneous SCC and marked pulmonary inflammation(10). A recently described model involved biallelic inactivation of LKB1 and PTEN in mouse lung leads to SCC that expresses the squamous markers keratin 5, p63, and SOX2(11). Chemopreventive interventions have been assessed in many of the murine preclinical models. This includes (but is not limited to): inhaled and systemic glucocorticoids; myoinositol; overexpression of prostacyclin synthase; dietary administration of the prostacyclin agonist iloprost; PPARγ overexpression; dietary administration of pioglitazone; COX inhibitors; the VEGF inhibitor vandetanib; and the anti-estrogen fulvestrant. The effect of COX inhibitors on lung cancer prevention has also been tested in murine models. It is likely the chemoprevention world will take a cue from lung cancer therapeutics by determining the altered pathways in specific premalignant lesions and employing targeted (or ‘precision’) chemoprevention in the next generation of trials. Reference List (1) Malkinson AM, Koski KM, Evans WA, Festing MF. Butylated hydroxytoluene exposure is necessary to induce lung tumors in BALB mice treated with 3-methylcholanthrene. Cancer Res 1997;57:2832-4. (2) Johnson L, Mercer K, Greenbaum D, Bronson RT, Crowley D, Tuveson DA, et al. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 2001;410:1111-6. (3) Regales L, Balak MN, Gong Y, Politi K, Sawai A, Le C, et al. Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors. PLoS One 2007;2:e810. (4) Malkinson AM. Primary lung tumors in mice: an experimentally manipulable model of human adenocarcinoma. Cancer Res 1992;52:2670s-6s. (5) Witschi H. Tobacco smoke as a mouse lung carcinogen. Exp Lung Res 1998;24:385-94. (6) Stearman RS, Dwyer-Nield L, Zerbe L, Blaine SA, Chan Z, Bunn PA, Jr., et al. Analysis of orthologous gene expression between human pulmonary adenocarcinoma and a carcinogen-induced murine model. Am J Pathol 2005;167:1763-75. (7) Wang Y, Zhang Z, Yan Y, Lemon WJ, LaRegina M, Morrison C, et al. A chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility. Cancer Res 2004;64:1647-54. (8) Hudish TM, Opincariu LI, Mozer AB, Johnson MS, Cleaver TG, Malkoski SP, et al. N-nitroso-tris-chloroethylurea induces premalignant squamous dysplasia in mice. Cancer Prev Res (Phila) 2012;5:283-9. (9) Keith RL, Miller YE. Lung cancer chemoprevention: current status and future prospects. Nat Rev Clin Oncol 2013;10:334-43. (10) Xiao Z, Jiang Q, Willette-Brown J, Xi S, Zhu F, Burkett S, et al. The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas. Cancer Cell 2013;23:527-40. (11) Xu C, Fillmore CM, Koyama S, Wu H, Zhao Y, Chen Z, et al. Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression. Cancer Cell 2014;25:590-604.
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MS14.02 - Clinical Trials: Who Are the Proper Cohorts and How Do You Recruit Subjects? (ID 1909)
14:15 - 15:45 | Author(s): S. Lam
- Abstract
- Presentation
Abstract:
Although chemoprevention as a means of reducing cancer incidence has been successful for basal cell carcinoma of skin, breast, and prostate cancer, after three decades of research, none of the phase III trials with agents such as Beta-carotene, retinol, 13-cis-retinoic acid, alpha-tocopherol, N-acetylcysteine, acetylsalicylic acid, or selenium have demonstrated beneficial and reproducible results in preventing lung cancer, likely due to the complexity of genomic alterations in lung cancer (1). Intermediate endpoint biomarkers such as bronchial metaplasia or dysplasia have been used in Phase II trials (2). Studies on the natural history of pre-neoplastic lesions in the central airways showed that patients with high-grade dysplasia or carcinoma in-situ are more likely to develop invasive cancer at the same or another site in the lungs than those with low grade lesions. However, pre-invasive bronchial lesions may be more of a marker of lung cancer risk because more cancers developed from a separate site in the same individual than progression from an initially biopsied dysplastic site and a significant proportion of the cancers are found by CT rather than by bronchoscopy (3,4). The advantage of using bronchial metaplasia/dysplasia for phase II chemoprevention trials is that these lesions can be localized and biopsied using white light and autofluorescence bronchoscopy for histopathology confirmation. However, with a steady decline in the prevalence of centrally located squamous cell carcinomas and a shift to adenocarcinomas which are usually located in the peripheral lung beyond the range of sampling by standard flexible bronchoscopes, it has become increasingly difficult to enrol participants with bronchial dysplasia for clinical trial. With the implementation of low dose computed tomography for screening of lung cancer, alternative intermediate endpoint biomarkers, such as CT detected non-calcified lung nodules are being investigated for phase II lung cancer chemoprevention trials (5). The limitations of using CT-detected lung nodules as an intermediate endpoint are the lack of confirmation of the underlying pathology and variable growth behaviour of sub-solid nodules especially non-solid nodules (6,7). Without confirmation of the pathology (atypical adenomatous hyperplasia versus adenocarcinoma in-situ versus minimally invasive or invasive adenocarcinoma), when a nodule is first seen, volume doubling time measurement is meaningless to determine malignant behaviour. Endoscopic optical imaging tools such as combined auto-fluorescence-optical coherence tomography (8) are promising methods to localize and characterize small peripheral lung lesions for tissue or liquid biopsy for pathological diagnosis and molecular characterization (Example shown in Figure 1). The availability of accurate lung cancer risk prediction models such as the one developed by Tammemagi and co-workers (9 ) opens the possibility of using lung cancer as the endpoint for chemoprevention trials in high risk ever smokers instead of intermediate endpoint biomarkers to test chemopreventive agents that have sound biological basis. Prospective evaluation of a prototype PLCOm2012 lung cancer risk prediction model in the Pan-Canadian early Detection of Lung Cancer Study showed that a 3 years lung cancer risk of ≥2% can reliably identify a sufficient number of ever smokers who will develop lung cancer within 3 years to make it feasible to use lung cancer incidence as the endpoint for chemoprevention trials with a sample size of approximately 2500 participants. The sample size can be significantly reduced by using a nodule malignancy risk prediction model (10). There is great potential to partner with lung cancer screening programs for developing and testing biologically rationalized agents for chemoprevention clinical trials within this framework, which may lead to their eventual implementation in screening programs to improve patient outcomes. Figure 1. Autofluorescence –Optical Coherence Tomography images of a CT-detected lung nodule showing the invasive and lepidic components of the adenocarcinoma confirmed by transbronchial lung biopsy. Figure 1 Supported by the Terry Fox Research Institute, Canadian Partnership Against Cancer, the Canadian Institute of Health Research and Lung Cancer Canada. References 1. Szabo E, Mao JT, Lam S, Reid ME, & Keith RL (2013) Chemoprevention of lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest 143(5 Suppl):e40S-60S. 2. Keith RL, Blatchford PJ, Kittelson J, Minna JD, Kelly K, Massion PP, et al. Oral iloprost improves endobronchial dysplasia in former smokers. Cancer Prev Res (Phila). 2011;4:793-802. 3. Ishizumi T, McWilliams A, MacAulay C, Gazdar A, Lam S. Natural history of bronchial preinvasive lesions. Cancer Metastasis Rev. 2010;29:5-14. 4. Jeremy George P, Banerjee AK, Read CA, O'Sullivan C, Falzon M, Pezzella F, et al. Surveillance for the detection of early lung cancer in patients with bronchial dysplasia. Thorax. 2007;62:43-50 5. Veronesi G, Szabo E, Decensi A, Guerrieri-Gonzaga A, et al. Randomized Phase II trial of inhaled budesonide versus placebo in high-risk individuals with CT screen-detected lung nodules. Cancer Prev Res 2011; 1:34-42. 6. Massion PP. Walker RC. Indeterminate pulmonary nodules: Risk for having or for developing lung cancer? Cancer Prev Res 2014;7:1173-1178. 7. Pinsky PF, Nath PH, Gierada DS, Sonavane S, Szabo E. Short- and long-term lung cancer risk associated with noncalcified nodules observed on low-dose CT. Cancer Prev Res (Phila). 2014;7:1179-85. 8. Pahlevaninezhad H, Lee AM, Shaipanich T, Raizada R, Cahill L, Hohert G, Yang VX, Lam S, MacAulay C, Lane P. A high-efficiency fiber-based imaging system for co-registered autofluorescence and optical coherence tomography. Biomed Opt Express. 2014 Aug 6;5(9):2978-87. doi: 10.1364/BOE.5.002978. eCollection 2014 Sep 1. 9. Tammemagi MC, et al. (2013) Selection criteria for lung-cancer screening. The New England journal of medicine 368(8):728-736. 10. Tammemagi MC, Lam S. Screening for lung cancer using low dose computed tomography. BMJ. 2014 May 27;348:g2253. doi: 10.1136/bmj.g2253. Review.
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MS14.03 - Pathologic Biomarkers of Risk and Benefit of Treatment (ID 1910)
14:15 - 15:45 | Author(s): D.T. Merrick
- Abstract
- Presentation
Abstract:
Prevention of lung cancer could lead to a significant reduction in the mortality associated with this disease. Identification of individuals at high risk for the development of invasive lung cancer is critical to establishing efficient and effective screening and prevention programs. The presence of premalignant lesions including atypical adenomatous hyperplasia/adenocarcinoma-in-situ (AAH/AIS) and bronchial dysplasia (BD), which represent precursors of adenocarcinoma and squamous cell carcinoma (SCC) respectively, provide targets that can be studied by histologic, radiographic and molecular techniques to define biologic characteristics that are indicative of risk and potential cellular activities that can be targeted for prevention. The histologic features of premalignant lesions have been well described in published WHO defined classification systems (1). Accurate histologic assessment of precursor lesions of lung adenocarcinoma prior to development of invasive cancer is limited by sampling considerations. To establish a diagnosis of premalignant AAH or AIS, the whole lesion must be examined and size criteria and histologic confirmation of lack of invasion must be documented. Because resection is generally restricted to cases of invasive cancer, tissue from AAH or AIS prior to development of invasive adenocarcinoma are rare, and most analyses of these lesions are performed on lesions that are associated with or occur as synchronous independent lesions of invasive cancer in resection specimens. However, a number of recent publications have begun to describe non-lesion associated biomarkers that can be correlated with radiographic features that appear to faithfully distinguish premalignant from invasive peripheral lung lesions. While AAH and AIS are considered to be precursors of adenocarcinomas derived from the terminal respiratory unit (TRU), a recently described premalignant lesion, mucous columnar cell change (MCCC), appears to be the precursor of a less common subset of adenocarcinomas derived from a region of the distal airways that is proximal to the TRU. This lesion has been reported to be present in up to 70% of the mucinous variant adenocarcinomas that are derived from these more central sites (2). This suggests that MCCC may be amenable to sampling at a pre-invasive stage by bronchoscopic means. In contrast, BD is detectable prior to development of invasive cancer by bronchoscopy but cannot be identified by radiographic examination. Higher rates of progression to invasive SCC and/or carcinoma-in-situ for lesions with higher grades of atypia have been suggested in a number of studies and meta-analyses (3). We have assessed the relationship between persistence of BD and risk for development of invasive lung cancer employing a numeric scoring system (1=normal; 2-7=increasing levels of precursor atypia; 8=invasive cancer). These analyses have shown that higher histologic scores on follow-up biopsies at specific sites within the airway of individuals sampled over time are associated with higher baseline histologic score, the presence of papillary angiogenic change, and current smoking status (4). Multivariable analyses including these parameters show that sites in subjects that develop SCC have mean histologic scores on follow-up biopsy that are 1.55 higher than those in patient’s without development of invasive lung cancer. On a per subject basis, the frequency of SCC was significantly increased in subjects that showed multiple sites of BD at baseline that persisted as or progressed to high grade dysplasia (moderate dysplasia or worse, histology score > 5). A 33% increase in risk for development of SCC is associated with every 10% increase in percent of sites that persist/progress to HGD corresponding to an overall hazard ratio of 17.14 (CI 2.4, 123.3) for multifocal persistent BD. These findings lend support to the importance of a field effect in lung carcinogenesis and suggest a potential role for histologic demonstration of persistent field change as an indicator of risk for the development of lung SCC. A number of biomarkers have been studied to determine their relationship with outcomes in premalignant lung lesions. Direct analyses of AAH and AIS have shown that a significant number of these lesions harbor the activating EGFR driver mutations seen in invasive adenocarcinoma of the lung. The potential of these mutational events to act as predictors of progression is under investigation, and a case report has demonstrated response to EGFR inhibitors of radiographically established multifocal premalignant disease in which an EGFR mutation was demonstrated in biopsy tissue of one of the lesions (5). Interestingly, the tumors associated with MCCC show a higher proportion of KRAS mutations. Non-lesional biomarkers of risk such as a recently reported assay measuring germline DNA repair activity that correlates decreased repair capability with increased risk for malignancy show promise for risk prediction (6). BDs, like SCC, demonstrate frequent genetic alterations in tumor suppressor genes and show characteristic associated alterations in gene methylation, loss of heterozygosity and gene copy number gains that have been associated with increased risk (7-10). In an analysis of a small series of cases in which sites with BD were observed to directly progress to invasive SCC, we have demonstrated frequent TP53 and some other mutations in precursor lesions. Furthermore, via pathway analysis of genes that we have found to be differentially expressed between persistent and regressive bronchial dysplasia, we have identified altered control of cell cycle, adhesion and immune activity (see abstract #3026) to be associated with persistence of BD. Overexpression of polo-like kinase 1 (PLK1) is the most prominent cell cycle control alteration associated with persistence and its role as a mediator of progression through the G2-M DNA damage checkpoint suggests a potential mechanism by which genomic instability can be promoted in high risk premalignant BD. PLK1 inhibitor treatment of primary cultures derived from sites of persistent BD causes an arrest of growth in S/G2 phase and induces apoptosis, neither of which occurs when PLK1 inhibitor is applied to primary cultures of normal bronchial epithelium. Histologic features and molecular biomarkers of premalignant lung lesions provide means by which risk can be assessed, appropriate targets for prevention can be identified and efficacy of preventive therapies can be measured. References 1. Travis WD, Brambilla E, Muller-Hermelink HK and Harris CC. Pathology and genetics: tumors of the lung, pleura, thymus and heart. World Health Organization Classification of tumours. Lyon: IARC; 2004. p. 9-124. 2. Weichart W and Warth A. Early lung cancer with lepidic pattern: adenocarcinoma in situ, minimally invasive adenocarcinoma, and lepidic predominant adenocarcinoma. Curr Opin Pulm Med 2014, 20:309–316 3. Ishizumi T, McWilliams A, Macaulay C, Gazdar A and Lam S. Natural history of bronchial preinvasive lesions. Cancer Metastasis Rev 2010;29:5-14. 4. Merrick DT, Haney J, Petrunich S, Sugita M, Miller YE, Keith RLet. al. Overexpression of vascular endothelial growth factor and its receptors in bronchial dysplasia demonstrated by quantitative RT-PCR analysis. Lung Cancer 2005;48(1):31-45. 5. Pastorino U, Calabro E, TamboriniE, MarchianoA, Orsenigo M, Fabbri A, Sozzi G, Novello S, and De Marinis F. Prolonged Remission of Disseminated Atypical Adenomatous Hyperplasia Under Gefitinib. J Thorac Oncol 2009;4: 266–267. 6. Sevilya Z, Leitner-Dagan Y, Pinchev M, Kremer R, Elinger D, Rennert HS, Schechtman E, Freedman LS, Rennert G, Paz-Elizur T, and Livneh Z. Low Integrated DNA Repair Score and Lung Cancer Risk. Cancer Prev Res; 7(4); 398–406. 7. Nakachi I, Rice JL, Coldren CD, Edwards MG, Stearman RS, Glidewell SC, Varella-Garcia M, Franklin WA, Keith RL, Lewis MT, Gao B, Merrick DT, Miller YE, and Geraci MW. Application ofSNPMicroarrays to theGenome-Wide Analysis of Chromosomal Instability in Premalignant Airway Lesions. Cancer Prev Res; 7(2); 255–65. 8. Massion, P., Zou, Y., Uner, H., Kiatsimkul, P.,Wolf, H. J., Baron, A. E., et al. Recurrent genomic gains in preinvasive lesions as a biomarker of risk for lung cancer. PLoS ONE 2009;4(6):e5611. 9. Wistuba, I. I., Behrens, C., Virmani, Ak, Mele, G., Milchgrub, S., Girard, L., et al. High resolution chromosome 3p allelotyping of human lung cancer and bronchial epithelium reveals multiple, discontinuous sites of 3pallele loss and three regions of frequent breakpoints. Cancer Res 2000;60:1949–1960. 10. Belinsky SA[1], Liechty KC, Gentry FD, Wolf HJ, Rogers J, Vu K, Haney J, Kennedy TC, Hirsch FR, Miller Y, Franklin WA, Herman JG, Baylin SB, Bunn PA, Byers T. Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. Cancer Res 2006; 66(6):3338-44.
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MS14.04 - Chemoprevention Clinical Trials: How Do We Move Forward? How Do We Identify Valid End Points? (ID 1911)
14:15 - 15:45 | Author(s): E. Szabo
- Abstract
- Presentation
Abstract:
The ability to intervene in the process of carcinogenesis is predicated on an understanding of the pathways leading to invasive cancer and availability of targeted tools to abrogate the resulting processes. Thus, effective chemoprevention has been hampered by the evolving understanding of lung cancer as a heterogeneous set of malignancies arising from a multitude of diverse molecular deregulations. The simplistic view that early intervention (before the evolution of multiple complex mutational events that are characteristic of tobacco-related malignancies) is more likely to be effective than late intervention has been replaced by the realization that many complex abnormalities actually do occur early and we simply do not understand which individual abnormalities or combinations of abnormalities would derail the inevitable progression to invasive and metastatic cancer. To assess efficacy, well designed clinical trials need to have end points that are informative. For phase III trials, the ideal end point would be cancer-related mortality so that cancer overdiagnosis does not cloud the issue. Realistically, cancer incidence is a more achievable and sufficiently informative end point. Phase II trials, however, depend on intermediate end points that are surrogates for cancer incidence, in a manner analogous to tumor shrinkage or progression-free survival being a surrogate for survival in phase II cancer treatment trials. Examples of end points that have been used in a variety of phase II chemoprevention trials are premalignant lesions, proliferative indices, and a variety of biomarkers of risk or malignant potential. It must be emphasized that to be useful, intermediate end points should be integrally involved in the process of carcinogenesis, differentially expressed in at-risk vs. normal epithelium, and modulated by effective interventions well above the level of spontaneous fluctuation (1). To date, no intermediate end point has been validated to replace lung cancer incidence, but such biomarkers can significantly inform drug development and decision-making for subsequent phase III trials. Nevertheless, the histologic evolution of squamous carcinogenesis, with progression from bronchial metaplasia through varying grades of dysplasia to carcinoma in situ is well described (2). This knowledge has allowed for clinical trials based on pre- and post-treatment assessment of effect of interventions on bronchial histology. However, the rate of progression of dysplasia to invasive cancer is variable even though high grade histologies are associated with higher rates of progression. Therefore, studies assessing dysplasia need to have placebo controls to correct for spontaneous and biopsy-induced regression. A recent trial of a prostacyclin analogue, iloprost, showed improvement in bronchial histology in former smokers after 6 months of treatment (3). These results will be extended in a soon-to-open trial of inhaled iloprost in a similar population and will include analyses of potential molecular predictors of histologic progression. Ongoing efforts are focusing on understanding the process of carcinogenesis by profiling premalignant lesions, both in a cross-sectional manner with regard to lesions identified at time of lung cancer resection (4) and with longitudinal follow-up. Understanding the natural history of premalignant lesions will help determine which ones progress, why they progress, and, therefore, which end points are likely to be most informative. An alternative way to approach the issue is to examine the at-risk epithelial field to identify biomarkers associated with progressive carcinogenesis. Gustafson et al. showed that the PI3K pathway is upregulated early during lung carcinogenesis and that an intervention with a drug, myo-inositol, that resulted in regression of bronchial dysplasia also inhibited PI3K activation in the histologically normal bronchial epithelium obtained by bronchial brushings (5). These data suggest that upregulated PI3K could potentially identify smokers at increased lung cancer risk and that pathway inhibition could serve as an end point for assessing treatment effect. This hypothesis is undergoing further testing in a recently finished phase II trial of myo-inositol in current and former smokers with dysplasia, where normal bronchial epithelium was collected and will be tested for PI3K activation pre- and post-treatment. Similarly, Spira et al. showed that gene expression classifiers from bronchial brushings of histologically normal epithelium obtained from individuals undergoing bronchoscopy for suspect lung cancer can aid in the diagnosis of lung cancer and can serve as lung cancer biomarkers (6,7). These classifiers have potential to be adapted to surrogate tissues further up in the aerodigestive tract, such as the nasal epithelium, and are being tested in chemoprevention ongoing clinical trials (8; NCT02123849). How do we move forward? A better understanding of the early carcinogenic processes and which processes are operative in individual persons is key to designing clinical trials that bring the prospect of precision medicine to lung cancer chemoprevention. The focus on a molecular understanding of premalignant lesions and the at-risk field is at the center of current efforts to identify informative end points for chemoprevention clinical trials. References Szabo E. Phase II cancer prevention clinical trials. Semin Oncol 2010;37:359-66. Saccomanno G et al. Development of carcinoma of the lung as reflected in exfoliated cells. Cancer 1974;33:256-70. Keith R et al. Oral iloprost improves endobronchial dysplasia in former smokers. Cancer Prev Res 2011;4:793-802. Ooi AT et al. Molecular profiling of premalignant lesions in lung squamous cell carcinomas identifies mechanisms involved in stepwise carcinogenesis. Cancer Prev Res 2014;7:487-95. Gustafson AM et al. Airway PI3K pathway activation is an early and reversible event in lung cancer development. Sci Trans Med 2010;2:26ra25. Spira A et al. Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer. Nat Med 2007;13:361-6. Silvestri GA et al. A Bronchial Genomic Classifier for the Diagnostic Evaluation of Lung Cancer. N Eng J Med 2015 May 17 [Epub ahead of print]. Zhang X et al. Similarities and differences between smoking-related gene expression in nasal and bronchial epithelium. Physiol Genomics 2010;41:1-8.
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Author of
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MINI 11 - Tobacco Control and Prevention (ID 108)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Prevention and Tobacco Control
- Presentations: 1
- Moderators:S.M. Janes, E. Stone, K.M. Cummings
- Coordinates: 9/07/2015, 16:45 - 18:15, 601+603
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MINI11.04 - A New Preclinical Model of Airway Progenitor Cells to Identify Responders to Iloprost-Mediated Chemoprevention (ID 1698)
16:45 - 18:15 | Author(s): Y.E. Miller
- Abstract
- Presentation
Background:
Lung cancer is the leading cause of cancer related deaths worldwide. The 5-year survival rate for this cancer is only 16%. Chemoprevention can improve prognosis in these patients. However, previous attempts at lung cancer chemoprevention that were soley based on epidemiological data were ineffective. Squamous cell lung cancer develops through a series of bronchial lesions or dysplasia. Persistent dysplasia harbors similar genetic changes as the tumor and has significantly higher chance of progression. Thus, bronchial dysplasia is a risk biomarker for SCC and improvement in dysplasia grade can be used as an outcome for chemoprevention trials. The long-acting prostacyclin analogue, iloprost is the only drug that has improved dysplasia in former smokers (p = 0.006). Despite this positive outcome we have little insight into the mechanisms of iloprost function. Understanding these mechanisms would be essential to identify people who have the highest chance to benefit from iloprost treatment. We propose that this endeavor will require a preclinical model that recapitulates the human disease and is amenable to mechanistic studies.
Methods:
Airway progenitor cells are critical for the maintenance of normal airways, because of their ability to self-renew (i.e. replicate) and differentiate into all cell-types of the airway (i.e. multipotentiality). Together these properties allow progenitors to return injured tissue to normal structure and function. In dysplasia, normal bronchial epithelium is changed into one that contains increased numbers of basal cells and lacks ciliated cells. These findings led to our hypothesis that ‘airway progenitors are malfunctioning in dysplasia’. Previously we showed that Keratin (K) 5/p63-expressing basal cells are the multipotential progenitors of the airway epithelium. During in vitro culture these cells form a unique 3-deimensional structure called the rim clone, which allows them to be distinguished from non-progenitors. To investigate a role of epithelial progenitors in dysplasia, we have collected bronchial biopsies from high-risk smokers and purified rim clone forming basal progenitor cells.
Results:
We demonstrate that both self-renewal and multipotentiality of progenitors is significantly (p < 0.001 for both) decreased in dysplasia. During differentiation in vitro at the air-liquid interface, progenitors from normal biopsies generated a normal epithelium. In contrast, progenitors from dysplasia made a squamous epithelium containing only basal cells and lacking ciliated cells. Mutational analyses of paired samples from epithelial brushings and biopsy-derived progenitors identified the same somatic mutations in p53, Notch 1, Notch 3, Survivin and FGFR1. Thus, epithelial progenitor culture reflects the histologic and genetic changes of dysplasia and therefore can be used as a personalized, preclinical model. A proof of concept study where dysplastic progenitor cells were treated with iloprost resulted in decreased dysplasia in 2 out of 3 cases.
Conclusion:
Thus our data indicate that progenitor cell cultures from a patient’s dysplasia may be used to identify responders versus non-responders to iloprost, as well as other chemopreventives. Future studies could focus on identifying downstream mechanisms via which iloprost exerts its beneficial effect.
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MINI 12 - Biomarkers and Lung Nodule Management (ID 109)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:J.M. Siegfried, H.I. Pass
- Coordinates: 9/07/2015, 16:45 - 18:15, 401-404
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MINI12.02 - Clinical Utility of Chromosomal Aneusomy in High Risk Individuals (ID 1299)
16:45 - 18:15 | Author(s): Y.E. Miller
- Abstract
- Presentation
Background:
In the context of CT screening in current and former smokers at high risk for lung cancer, the false positive rate is high (26% at first NLST screening; 13% with Lung-RADS criteria applied to NLST) and indeterminate nodules are frequently discovered. Noninvasive biomarkers are urgently needed to reduce false positives with screening CT and to improve risk stratification in those with indeterminate nodules. The Colorado (CO) Lung SPORE program performed a retrospective longitudinal evaluation (Pepe Phase 3 validation) to assess the potential of chromosomal aneusomy detected in sputum via fluorescence in situ hybridization (CA-FISH) as a biomarker for early detection in four nested case-control studies. Two of the cohorts (ACRIN/NLST and PLuSS) enrolled current and former smokers to investigate use of low dose CT to diagnose lung cancer. The other two were Colorado cohorts in which pulmonary clinic patients (mostly current and former smokers) were enrolled to investigate biomarkers to predict lung cancer. One of these cohorts (CO High Risk) was a COPD population and the other, still in the accrual phase, comprises patients referred for care of indeterminate lung nodules (CO Nodule).
Methods:
The cohorts were grouped into a Screening cohort (ACRIN/NLST (49 cases, 96 controls) and PLuSS (48 cases, 89 controls)) and a High Risk cohort (CO High Risk (55 cases, 59 controls) and CO Nodule (13 cases, 10 controls)). The CA-FISH assay was a 4-target panel including genomic sequences encompassing the EGFR and MYC genes, and the 5p15 and centromere 6 regions or the FGFR1 and PIK3CA genes. At the subject level, the assay was scored on a 4-category scale representing normal, probably normal, probably abnormal and abnormal. Operating characteristics (with 95% CI) of the assay were estimated for each group of cohorts overall and separately for COPD patients: sensitivity, specificity, likelihood ratio+ (LR+) and likelihood ratio- (LR-).
Results:
Using the cutoff of abnormal vs. not abnormal for CA-FISH, sensitivity and specificity for Screening subjects are 0.20 (0.13, 0.30) and 0.84 (0.78, 0.89), respectively; and for High Risk subjects are 0.67 (0.55, 0.78) and 0.94 (0.85, 0.98), respectively. Likelihood ratios for Screening subjects are LR+: 1.36 (0.81, 2.28) and LR-: 0.93 (0.83, 1.05), and for High Risk subjects are LR+: 11.66 (4.44, 30.63), and LR-: 0.34 (0.24, 0.48). Similar results were observed when only COPD subjects were analyzed.
Conclusion:
The high LR+ of sputum CA-FISH indicates that this noninvasive biomarker could be a clinically useful adjunct to CT among patients in high risk settings. Whether this same high level of LR+ will be reproducible in patients at high risk because of their indeterminate nodules remains to be seen. If so, a hypothetical patient with indeterminate nodules and a pre-test (CA-FISH) lung cancer risk of 20% would have a post-test probability of lung cancer of 78% if the CA-FISH test were positive. In the screening setting, however, the low LR+ of CA-FISH limits its clinical utility. Prospective assessment of sputum CA-FISH is ongoing in the Nodule Cohort of the CO Lung SPORE.
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MINI 22 - New Technology (ID 134)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
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MINI22.10 - A New Approach to Large Scale Proteomic Profiling to Uncover Tumor Phenotypes (ID 2166)
16:45 - 18:15 | Author(s): Y.E. Miller
- Abstract
- Presentation
Background:
Genomic profiling is a powerful method for identifying mutations that drive tumors and matching patients to targeted therapies. However, this may only be a transient solution and resistance commonly emerges as the mechanism of targeted inhibition is overcome. Proteomic profiling of the tumor provides a dynamic tool to survey altered protein expression and deregulated pathways, which in turn may implicate specific treatments or identify novel therapeutic targets. Mass spectrometry offers highly multiplexed proteomic measurements, but extensive sample pre-processing and low sample throughput can lead to extended analysis times of weeks or months. Thus a need exists for a high throughput, sensitive and quantitative platform for proteomic analysis.
Methods:
We used the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV, to analyze protein lysates from 63 lung tumor samples. The assay does not require sample pre-fractionation, and this study (which generated over 142,000 protein measurements) represents less than one day of SOMAscan throughput. The study consisted of matched tumor/non-tumor protein lysates prepared from 18 squamous cell carcinoma and 45 adenocarcinoma fresh-frozen resected specimens, 86% of which were Stage I/II. The paired log~2~ tumor/non-tumor ratio was calculated and hierarchical clustering heat maps and dendrograms were constructed to identify related protein regions and tumor phenotypes.
Results:
Common proteomic changes and unique tumor phenotypic groups were identified by unbiased clustering algorithms. Large, consistent tumor/non-tumor differences of at least 4-fold were observed for 35 proteins in at least 20 (32%) of the tumors. Some of these proteins were more than 100-fold higher in individual tumors. The two most commonly elevated proteins were thrombospondin 2 and MMP12, which were increased in 81% and 61% of the tumors, respectively. We have previously reported higher levels of MMP12 in the serum of lung cancer patients, and the current data supports a tumor-associated origin for circulated MMP12. A second analysis identified sub-phenotypes of tumors clustered by common protein alterations independent of histological classification or mutation status. Many of these tumor subsets had increased expression of known oncology drug targets.
Conclusion:
Broad, unbiased high-throughput proteomic profiling of tumor tissue may reveal individual phenotypes that hold the potential to respond to targeted therapies and to monitor therapeutic efficacy throughout treatment. Measuring proteins complements mutation analysis by enabling therapeutic selection beyond driver mutation targets, including immune modulator therapies, repurposing existing drugs and enriching clinical trials with likely responders. While genomics is a fixed snapshot, blood- and tissue-based serial proteomic measurements respond to change and can lead to the personalized adaptation of treatment and identification of novel therapeutic targets.
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MINI 23 - Lung Cancer Risk: Genetic Susceptibility and Airway Biology (ID 135)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Screening and Early Detection
- Presentations: 2
- Moderators:P.E. Postmus, R. Young
- Coordinates: 9/08/2015, 16:45 - 18:15, 401-404
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MINI23.06 - Discussant for MINI23.01, MINI23.02, MINI23.03, MINI23.04, MINI23.05 (ID 3424)
16:45 - 18:15 | Author(s): Y.E. Miller
- Abstract
- Presentation
Abstract not provided
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MINI23.10 - Subtraction of Allelic Fractions (Delta-θ): A Sensitive Metric to Detect Chromosomal Alterations in Heterogeneous Premalignant Specimens (ID 2434)
16:45 - 18:15 | Author(s): Y.E. Miller
- Abstract
- Presentation
Background:
Lung squamous carcinoma is believed to arise from premalignant bronchial epithelial dysplasia, which demonstrates progressive histologic changes leading up to invasive cancer. However, only a small subset of these lesions progress to carcinoma. Recent studies have shown that somatic chromosomal alterations (SCAs) status is a better biomarker than premalignant histology alone. Single-nucleotide polymorphism microarray (SNP array) has been frequently used to delineate these genomic alterations across the whole genome. However, the cellular heterogeneity, from clinical samples such as endobronchial specimens, is a basic obstacle to perform sensitive and accurate detection of SCAs.
Methods:
We used: 1) a lung cancer cell line (NCI-H1395) and its matched lymphoblastoid (NCI-BL1395) cell line; 2) frozen lung tissues containing different percentage of invasive cancer cells surgically resected from a patient; and 3) biopsies and brushings obtained at the visually concerning areas during bronchoscopy. The histology of the clinical samples were graded by the study pathologist. Genomic DNA was isolated from each sample, quantified, and labeled for Illumina SNP array (HumanOmni 2.5-Quad BeadChip). Data analysis and visualizations were performed using Partek Genomic Suite 6.6 software.
Results:
Our study focused on the detection of SCAs by the comparison of genomic DNAs from cancer/premalignant cells (subject) to blood/normal cells (reference) from the same individual. We tested a B allele frequency metric, the subtraction of allelic fractions (delta-θ), on a standardized mixture of genomic DNAs from a lung cancer cell line and its matched lymphoblastoid cell line. Delta-θ proved to be a sensitive parameter to clearly delineate SCAs present in the tumor cell line even with a large proportion of normal cells (up to 90%). To explore the utility of using delta-θ for heterogeneous samples, we used clinical lung cancer specimens with known cancer cell content. In comparison to the other publicly available analytical metrics/algorithms (conventional Log R Ratio plot, mirrored B Allele Frequency plot, and GAP algorithm), delta-θ performed as well or better (with lower computational power needed), and enabled the detection of SCAs even in highly heterogeneous clinical samples (<30% tumor cell content). In addition, we completed a study using a number of bronchial biopsies and brushings with histologic grade ranging from normal to squamous cell carcinoma. SCAs were rarely detected in those of low to mild dysplasia, while they were detected in approximately 25% of moderate or severe dysplasia, and in all carcinoma in situ (CIS) and squamous cell carcinoma specimens. Longitudinal, repeated samplings from a high risk patient who persistently showed high grade dysplasia across the bronchus, revealed that delta-θ could identify SCAs continuously across the whole genome. The fact this individual had highly overlapping SCAs between different bronchial locations indicates genomic field cancerization may occur, along with the histological field effect in premalignant epithelium.
Conclusion:
In SNP microarray studies, delta-θ is a highly sensitive metric for detecting SCAs even in heterogeneous dysplastic bronchial specimens. SNP array may be a powerful tool to understand premalignant genetic alterations and field cancerization.
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ORAL 23 - Prevention and Cancer Risk (ID 121)
- Event: WCLC 2015
- Type: Oral Session
- Track: Prevention and Tobacco Control
- Presentations: 2
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ORAL23.02 - Pioglitazone for the Chemoprevention of Lung Cancer (ID 2419)
10:45 - 12:15 | Author(s): Y.E. Miller
- Abstract
- Presentation
Background:
Prior clinical studies have shown that the oral prostacyclin agonist iloprost improves bronchial dysplasia in former smokers. Prostacyclin is a PPAR gamma agonist, and epidemiologic and pre-clinical studies suggest PPAR gamma agonists like pioglitazone may chemoprevent lung cancer. Based on these promising results, a double-blind, placebo controlled, phase II trial of pioglitazone in subjects at increased risk for lung cancer was sponsored by the Department of Veterans Affairs.
Methods:
Subjects were selected for the trial if they met one the following criteria: current or former smoker (> 10 pack years); biopsy proven endobronchial dysplasia; airflow obstruction (FEV1/FVC < 0.70); or at least mild sputum cytologic atypia. Fluorescent bronchoscopy was performed with biopsy of 6 standard endobronchial sites and all other abnormally appearing areas. Subjects also had pulmonary function testings and quantitative high resolution CT scans at the start and completion of the trial. Subjects were then randomized to oral pioglitazone or placebo for 6 months and then a second fluorescent bronchoscopy with repeat biopsy of all the central airway areas sampled on the first bronchoscopy. The endobronchial biopsies were scored on a 1-8 scale based on WHO criteria. The primary endpoint for the study is change in maximum (worst) endobronchial histology.
Results:
A total of 90 subjects (46 pioglitazone, 44 placebo) have been enrolled in the trial, with 76 completing both bronchoscopies. Subjects are well matched in terms of age, gender, tobacco exposure, and sputum cytology. No significant differences in lung function were observed between the treatment groups. While the investigators remain blinded in regards to treatment group, aggregate data is available. Overall, mild dysplasia or worse was seen in 26% of the initial biopsies. Similar to prior studies, current smokers exhibited more dysplasia at baseline compared to former smokers (32.4% vs. 16.6%) and also had more angiogenic squamous dysplasia (11.7% vs. 3.2%). Our primary endpoint is change in maximum histology, and histologic scores from matched biopsies in all participants showed a change of at least 1 grade in 50.2% (25.9% improved, 24.3% progressed). More histologic changes were observed in current smokers (59.2%) than former smokers (41.7%). Summary data for the non-normal biopsy pairs (ie those with a histologic score of at least 2 on baseline biopsy) showed that the majority of pairs (73.7%) changed by at least one grade. Current smokers exhibited more progression (29.3%) compared to former smokers (14.6%).
Conclusion:
The pioglitazone lung cancer chemoprevention trial is currently in progress. The treatment has been well tolerated and histologic changes were observed in many of the subjects.
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ORAL23.03 - Role of Inflammatory Infiltrates in Promoting Persistence or Regression of Bronchial Dysplasia (ID 3026)
10:45 - 12:15 | Author(s): Y.E. Miller
- Abstract
- Presentation
Background:
Inflammatory infiltrates show differing capacities to eliminate malignant cells. This capacity is related to the polarization of key inflammatory cells in tumor infiltrates. A pathway analysis of genes that are differentially expressed between persistent and regressive bronchial dysplasia (BD) identified 13 pathways associated with persistence of which 8 were related to inflammation. We have hypothesized that differences in inflammatory infiltrate polarization may contribute to lung carcinogenesis and have employed gene expression and in situ analyses to characterize differences in inflammatory infiltrates related to persistence and regression of pre-malignant BD.
Methods:
Normalized gene expression levels (Affymetrix Hu 1.0) of selected genes related to inflammatory cell polarization features were analyzed to find differences associated with follow-up histology for BD. Validational analyses of these relationships were undertaken in studies of baseline biopsies selected to represent persistent (n=43) and regressive BD (n=39). These biopsies were analyzed by quantitative immunohistochemistry and dual immunofluorescence studies to characterize the overall proportion of subsets of T-lymphocytes and macrophages in each of the groups. Image analysis tools (Aperio) were used to characterize the density of inflammatory cell subsets in the stromal and epithelial compartments of biopsy tissue within defined areas.
Results:
Analysis of expression levels for a subset of inflammatory cell related genes assessed in a global gene expression analysis indicated significantly higher levels of expression of macrophage M1 markers HLA-DRA (p=0.01) and inducible nitric oxide synthetase (iNOS; p=0.02) and T-helper lymphocyte marker CD4 (p=0.04) in regressive BD compared to persistent BD. There was also a trend toward higher expression of cytotoxic T-lymphocyte marker CD8 in regressive BD (p=0.25). Expression of B-lymphocyte and neutrophil markers were not different between regressive and persistent BD. CD68 immunohistochemical stains (IHC) demonstrated a trend toward an increase in macrophages per area of combined dysplastic epithelium and underlying stroma with a mean increase in IHC positivity of 1.75-fold in regressive versus persistent BD (p=0.08). CD4 and CD8 IHC showed 1.36- and 1.19-fold increases, respectively, in regressive BD but these changes were not statistically significant (p=0.36 and p=0.43 respectively). Dual immunofluorescence was undertaken to determine if polarization specific subsets of macrophages correlated with regression or persistence of BD. Analysis of a preliminary subset of regressive (n=3) and persistent (n=3) BD demonstrates a wide range of M1 to M2 ratios (range = 0.84 – 4.82 for ratio of HLA-DRA-CD68 dual positive M1 to CD206-CD68 dual positive M2 macrophages per high power field, 400X). Additional analyses of macrophages are ongoing to determine if the polarization status is related to regression or persistence of BD, and analysis of markers of T-helper lymphocyte subsets are planned.
Conclusion:
Gene expression analyses indicate that increased expression of markers of M1 macrophages and T-helper lymphocytes are associated with regression, and in situ analyses suggest that differences in the amount of inflammatory cell subsets may be related to outcome in BD. These studies could have implications for predicting the behavior of premalignant disease and manipulating inflammatory activity in preventing progression of BD to invasive lung cancer.
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P2.06 - Poster Session/ Screening and Early Detection (ID 219)
- Event: WCLC 2015
- Type: Poster
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.06-007 - A miRNA Signature Derived From Independently Replicated Biomarkers of Non-Small Cell Lung Cancer (ID 1728)
09:30 - 17:00 | Author(s): Y.E. Miller
- Abstract
Background:
miRNAs have shown exceptional promise as biomarkers of lung cancer; however, no miRNA signatures have yet reached the clinic. Towards developing a signature with a high likelihood of being validated externally for clinical use, we screened a panel of 50 miRNAs shown to be effective biomarkers in at least two previous studies for distinguishing human lung cancer samples from non-cancer samples.
Methods:
Sixty tumor-normal pairs (33 adenocarcinoma, 27 squamous cell carcinoma) were used to identify the best-performing combination of 4 miRNAs for distinguishing tumor samples from normal. The miRNA levels were measured by RT-qPCR using Taqman custom-made microfluidics cards and primer pools purchased from Life Technologies. All possible combinations of 4 miRNAs were tested, and best performance was defined as the highest median area-under the receiver operating curve (AUC) obtained from 1000 bootstrap replicates. A second, independent set of 68 tumor-normal samples (half adenocarcinoma, half squamous) was used as a test set, and bootstrapping was used to determine the 95% confidence interval for the AUC.
Results:
The median AUC for the top-performing panel of 4 miRNAs in our training set was 0.96. Several other miRNA combinations exhibited AUCs > 0.95 as well. In our test set, the top-performing panel (and only panel tested) exhibited an AUC of 0.97 (0.93, 0.99). This panel consisted of miRs 26a, 145, 183 and 486. miRs 145 and183 have previously been shown, when used individually, to be significant lung tumor biomarkers in at least 4 previous studies; miR-486 has been replicated 8 times.Figure 1
Conclusion:
Consistent with previous studies, we’ve identified a panel of 4 miRNAs that shows excellent potential for diagnosing lung tumors. Each of these miRNAs has been replicated as a biomarker of lung cancer in at least two previous studies, suggesting a high likelihood of achieving clinical validation. Several previous studies have also shown that these four miRNAs are potentially useful as biomarkers for diagnosing lung cancer using blood samples, and we are currently pursuing such validation studies.
